There could be several reasons for you not detecting the peak of dopamine in an HLC EC detection procedure from the medium. I would suggest that your detection limit (sensitivity of the detction of the EC detector) to be increased while injecting your samples prepared from the medium. It also depends on when you are sampling the medium. always run a control medium sample. Add the sampled medium to an ice-cold deproteinization solution and after mixing keep it for at least 20 minutes for complete precipitation of proteins. Presence of protein in the samples injected, could cause thin layer of proteins to be fomed on the surface of the working electrode severely hampering the detection sensitivity, since oxidation of the catechole does not happen as the electrical potential applied will not reach oxidizable molecules in the samples.

Always run an appropriate amount (may be in femto mole concentration) of dopamine sample of dopamine before and after the run of a sample, to get the correct value. And try to inject not more than 20 microlitre. By the by, it would have been best if I knew more about your mobile phase. Good luck

Mohanakumar KP

When I measured dopamine from brain tissue samples, I used to keep my samples on ice to prevent the degradation of dopamine.

If you want to monitor dopamine release from cell kept in culture, I would suggest to put MAO inhibitor and/or DAT inhibitor in your supernatant.

Do you have ascorbic acid in your cell culture medium? This may help prevent oxidation. Also, try including 0.1% sodium meta-bisulfite in your supernatant samples as an antioxidant.

For dopamine detection in PC12 I follow procedure published from Yamboliev et al. (Eur J Neurosci. 2009 Sep;30(5):756-68): "Briefly, 115-μl aliquots from the samples were acidified with 3 μl 1 M perchloric acid to pH 2.6 and injected..."

i used 1N perchloric acid. Anyway you can also try to concentrate your medium with specific columns befor HPLC detection

Because of its catechol function dopamine is readily oxydized. Heavy metal ions appear to play an important role, that's why EDTA in the medium is of paramount importance. But make sure that your EDTA is not fully taken away by the complexation of existing metal ions. Best is to add an anti-oxidant as well. We successfully used ascorbid acid (0,11mM) in combination with EDTA (27uM). J Pharm Pharmacol 1983;35:786-92. Good luck!

Are you seeing any oxidation or degradation products in your HPLC? Even if you are losing signal for DA itself you should be able to track conversion products in the HPLC in order to test different extracellular media.

Ascorbic acid is a good antioxidant to protect DA; however, it also prevents the oxidation process during the electrochemical detection. You have to be aware of this issue. For this reason, some people prefer to use other acids such perchloric acid or HCl for ECD.

You should also consider (this is particularly important for a low DA concentration in your media) that any metal in contact with your solution would promote oxidation of your precious DA. You should try using PEEK materials or at least passivate your HPLC system before run your samples (your syringe also count). It can be done treating your HPLC (no column attached to your system) with the following sequence: 30 min water - 30 min methanol 100% - 30 min water - 30 min nitric acid 1M - 30 min water - 30 min disodic EDTA - 30 min water (flow rate 1 ml/min is fine). Then, flush your system with your mobile phase before reinstall your column and detection system back.

Hope it helps...

Ascorbic acid won't prevent the oxidation of dopamine if the voltage on the ECD is high enough. ECD is typically used for the detection of ascorbic acid, and 600 mV is high enough to cause oxidation of ascorbate, urate, and most anything else we throw in there (concentration dependent, of course).

I would like to point out that EDTA-metal complexes are not redox inactive like many people assume. In certain circumstances, like with large amounts of iron or copper contamination, EDTA can promote oxygen reduction to superoxide - especially with a convenient source of electrons, like ascorbic acid. The chelator of choice would be something that could ideally bind iron or copper in a way that redox activity is completely inhibited. We have successfully used DTPA in this regard.

Since you have perchloric in there, I agree that ascorbate and sodium meta-bisulfite might help prevent oxidation. The extracellular concentrations in vivo are generally quite high, so you probably could add more. Also-- what is the concentration of Ca++ ions in the medium... perhaps that is too low.

I agree with Tiziana Pascucci answer.

Thanks everyone for the responses. I really appreciate it.

Francois, Iuvone and Joop - I will try adding ascorbic acid or sodium metabilsulfite on my column. I thought EDTA would take care of the oxidation and hence I did not add additional antioxidants.

Tiziana - Thanks for the link. I will look into that.

Damiana - Unfortunately we cannot use the columns to concentrate the samples as they are super expensive and I generate a lot of samples per experiment.

Paul - I am seeing a loss of DA signal in my samples more than I can account for. So I do feel that I am losing fraction of my DA signal to oxidation which is not being detected on the column. Even the conversion products are not high enough everytime to analyze them. But I have not carefully looked into HVA or DOPAC till now. So, I will check that.

Miguel - Thanks for the insight. The column I use belongs to another lab. But I will try to get the washes done if ascorbic acid or sodium metabisulfite dont work.

Alexander - You have an excellent point. I will buy some DTPA in case the regular anti-oxidants dont work.

John - The concentration of CaCl2 in Hepes buffered saline solution is 2.6 mM. I collect the DA for 15 mins in this salt solution. Do you think I should increase it further?

Thanks a lot, everyone. I think I have enough leads to trouble shoot. I will keep you updated on the things I try. The PC12s grow slowly so it takes time for me to collect enough for my assay.

1. Try adding ascorbic acid and sodium metabisulfite with EDTA to the solutions.

2. If #1 does not work, try adding DTPA instead of EDTA.

3. If #1 and #2 dont work, I will try to wash the line without the column to free it from any possible iron/copper contamination.

It is interesting how the iron levels in the water here in Indiana are very high. However, MilliQ water should take care of that.

Hi,

I think most people that do microdialysis would say that 2.4-2.6 mM Ca++ is fine. However, if a detection problem remains, you might want to go higher, e.g., 3.0 or 3.2 mM. Probably most people would say that would be higher than physiological levels, but on the other hand, your conditions are not exactly physiological anyway. Just a thought.

There could be several reasons for you not detecting the peak of dopamine in an HLC EC detection procedure from the medium. I would suggest that your detection limit (sensitivity of the detction of the EC detector) to be increased while injecting your samples prepared from the medium. It also depends on when you are sampling the medium. always run a control medium sample. Add the sampled medium to an ice-cold deproteinization solution and after mixing keep it for at least 20 minutes for complete precipitation of proteins. Presence of protein in the samples injected, could cause thin layer of proteins to be fomed on the surface of the working electrode severely hampering the detection sensitivity, since oxidation of the catechole does not happen as the electrical potential applied will not reach oxidizable molecules in the samples.

Always run an appropriate amount (may be in femto mole concentration) of dopamine sample of dopamine before and after the run of a sample, to get the correct value. And try to inject not more than 20 microlitre. By the by, it would have been best if I knew more about your mobile phase. Good luck

Mohanakumar KP

I'm wondering if DA might undergo degradation during the extra processing required to extract the supranatent. Are you keeping the samples chilled during homogenizing and centrifuging? I like to spin at 14,000g for 30-45 min at 4C. It should be possible to measure your signal loss if you spike a handful of samples with a known DA concentration prior to processing (much much higher than you would see at physiological levels). Then you can compare the measured DA levels in your supranatent to what you get when you run a standard at that concentration. That should determine if your DA is somehow being lost during processing or not being picked up by the HPLC, due to tissues with mobile phase, electrodes, etc. Oh HPLC-ED! As a wise man once said, "Treat the HPLC with great respect. It knows what you are thinking and will punish you accordingly" Good luck!

I think that threason is that because of its catechol function dopamine is readily oxydized. Heavy metal ions appear to play an important role, that's why Ethylene diamminne tetraacetic acid in the medium is of paramount importance.

Look into the following article: The use of microwave irradiation with low formalin concentrations to enhance the conversion of dopamine into norsalsolinol in rat brain: a pilot study. C F Van Valkenburg, E Marani, M E Boon, P Visser

The Histochemical Journal 01/1990; 22(6-7):353-7.